A new crustin is involved in the innate immune response of shrimp Litopenaeus vannamei.

Crustin is an antimicrobial peptide (AMP) that plays a key role in the innate immunity of crustaceans. This study cloned a new crustin from Pacific white shrimp Litopenaeus vannamei, which we designated as LvCrustinB, using rapid amplification of cDNA ends (RACE). The full-length cDNA of LvCrustinB is 751 bp with an open reading frame (ORF) of 591 bp encoding a peptide of 196 amino acids that includes a putative signal sequence. LvCrustinB is a type II crustin that has a glycine-rich region and a single whey acidic protein domain (WAP) domain. The mRNA transcript of LvCrustinB was detected in all examined tissues and was found to be most abundantly expressed in the epithelium and muscle. The expression of LvCrustinB in hemocytes was significantly upregulated after L. vannamei was challenged with LPS, Vibrio parahaemolyticus, and white spot syndrome virus (WSSV). When LvCrustinB was knocked down with RNAi, the mortality rate of L. vannamei significantly increased after V. parahaemolyticus or WSSV infection. Recombinant LvCrustinB was produced using Pichia pastoris GS115 and was shown to bind to 2 g-positive bacteria (Staphylococcus aureus and Bacillus subtilis) and 2 g-negative bacteria (Escherichia coli and V. parahaemolyticus) via polysaccharides, which included PGN, LTA, and LPS. In vivo, the recombinant LvCrustinB remarkably protected L. vannamei from V. parahaemolyticus infection. These results suggest that LvCrustinB plays an important role in innate immunity and may be potentially utilized as antibacterial agents in shrimp.

Li M ，Ma C ，Zhu P ，Yang Y ，Lei A ，Chen X ，Liang W ，Chen M ，Xiong J ，Li C ... -  《-》

Molecular cloning, expression, promoter analysis and functional characterization of a new Crustin from Litopenaeus vannamei.

Antimicrobial peptides (AMPs) are the most important players in the innate immune system, providing a principal first-line of defense against the invading pathogens. Crustin, a type of whey acidic protein (WAP) domain-containing and cationic cysteine-rich AMP, can function in a protease inhibition or an effector molecule manner. In the present study, a new Crustin was cloned and identified from Pacific white shrimp Litopenaeus vannamei and designated as LvCrustinA. The full-length cDNA of LvCrustinA was 687 bp, with a 519 bp open reading frame (ORF) that encoded a peptide of 172 amino acids. Domain analysis indicated that LvCrustinA contained a Glycine-rich region in the N-terminal and a single WAP domain within eight cysteines in the C-terminal. The 5' upstream regulatory sequence of 1249 bp (promoter) was obtained using a genome walking method, and it contained several conserved transcription factors binding motifs including NF-κB, AP-1 and STAT (Signal transducers and activators of transcription). Dual-reporter assay showed that NF-κB transcription factors LvDorsal and LvRelish, and AP-1 transcription factor Lvc-Jun could up-regulate the promoter activity of LvCrustinA, suggesting that NF-κB and JNK-c-Jun pathways could be involved in regulating the expression of LvCrustinA. Moreover, LvCrustinA was abundantly expressed in immune related tissues such as gill, hemocyte and epithelium, and its expression was up-regulated in response to Vibrio parahaemolyticus and White spot syndrome virus (WSSV) challenges in gill tissue, suggesting that LvCrustinA could be involved in the host defense against bacterial and viral infection. Additionally, RNAi mediated knockdown of LvCrustinA resulted in shrimps with the higher cumulative mortality during V. parahaemolyticus and WSSV infection. Taken together, these results provided some insight into the expression and transcriptional regulatory role of LvCrustinA, and its defensive role against pathogenic infection.

Li M ，Ma C ，Li H ，Peng J ，Zeng D ，Chen X ，Li C ... -  《-》

Litopenaeus vannamei CK2 is involved in shrimp innate immunity by modulating hemocytes apoptosis.

Protein kinase CK2 (CK2) is a ubiquitous serine/threonine kinase with multiple cellular functions in vertebrates including apoptosis, differentiation, proliferation, survival, tumorigenesis, signal transduction, immune regulation and inflammation. In the current study, the catalytic and regulatory subunit homologs of Litopenaeus vannamei protein kinase CK2 (LvCK2α and LvCK2β) were cloned and characterized. LvCK2α has a full-length cDNA sequence of 1764 bp with a 1053 bp open reading frame (ORF) encoding a putative protein of 351 amino acids, which contains a typical serine/threonine kinase domain. On the other hand, LvCK2β has a 1394 bp full-length cDNA with an ORF of 663 bp encoding a protein with 221 amino acids, which contains a Casein kinase II regulatory subunit domain. Sequence and phylogenetic analysis revealed that LvCK2 was evolutionary related with the CK2 of invertebrates. Quantitative reverse transcription PCR (RT-qPCR) analysis showed that LvCK2α and LvCK2β transcripts were widely expressed in all shrimp tissues tested, and were both induced in hemocytes and hepatopancreas upon challenge with Vibrio parahaemolyticus, Streptoccocus iniae, lipopolysaccharide (LPS), and white spot syndrome virus (WSSV), suggesting their involvement in shrimp immune response. Moreover, RNA interference (RNAi) of LvCK2α resulted in increased hemocytes apoptosis, shown by high caspase 3/7 activity, increased number of apoptotic cells, coupled with an elevation in transcript levels of pro-apoptotic LvCaspase3 and LvCytochrome C, and a reduction in mRNA levels of pro-survival LvBcl2, LvIAP1, and LvIAP2. In addition, LvCK2α knockdown followed by V. parahaemolyticus challenge resulted in higher cumulative mortality of shrimp. Taken together, our current findings suggest that LvCK2 modulates shrimp hemocytes apoptosis as part of the innate immune response to pathogens.

Feng Q ，Huang Y ，Yao D ，Zhu C ，Li S ，Ma H ，Aweya JJ ，Zhang Y ... -  《-》

Characterization of a gill-abundant crustin with microbiota modulating function in Litopenaeus vannamei.

Gills as the respiratory tissue of crustacean encounter various threats in the environment. The immune defense in gills is essential to the health of shrimp. In the present study, we identified a novel gill-abundant crustin, LvCrustin Ⅱ-1, from the shrimp Litopenaeus vannamei. The full-length open reading frame of LvCrustin Ⅱ-1 is 522 bp, which encodes 173 amino acid residues. The amino acid sequence of LvCrustin Ⅱ-1 contained a signal peptide, a glycine rich region, a cysteine rich region and a whey acidic protein (WAP) domain. The expression level of LvCrustin Ⅱ-1 was significantly up-regulated at different time points after Vibrio parahaemolyticus immersion. When LvCrustin Ⅱ-1 was silenced by dsRNA interference, the count of bacteria colonies increased significantly in the hepatopancreas of shrimp after V. parahaemolyticus immersion, which indicated that the infection progress of pathogenic bacteria was accelerated after LvCrustin Ⅱ-1 knockdown. Compared with the microbiota of seawater, the lower proportion of aquatic bacteria and higher proportion of symbiont in the gills microbiota of shrimp indicated the bacterial colonization was modulated by the host. Knockdown of LvCrustin Ⅱ-1 changed the proportion of some potential pathogens and aquatic bacteria, which supported the idea that the new identified crustin in the gills played important roles in modulation of the microbiota community in shrimp. The present data provided new insights into the multiple functions of crustin in the immunity of shrimp.

Lv X ，Li S ，Yu Y ，Zhang X ，Li F ... -  《-》

The crustin-like peptide plays opposite role in shrimp immune response to Vibrio alginolyticus and white spot syndrome virus (WSSV) infection.

Crustin is an antimicrobial peptide (AMP) that plays a key role in innate immunity of crustaceans. In this study, we cloned the entire 660 bp crustin-like sequence with a 507 bp open reading frame encoding a 168 amino acid from Marsupenaeus japonicus. The crustin-like gene was primarily expressed in gills and over-expressed in shrimp hemocytes after challenge with WSSV or Vibrio alginolyticus. After knockdown crustin-like gene using specific double-stranded RNA (CRU-dsRNA), IMD, Rab7, L-lectin, mitogen-activated protein kinase, p53, prophenoloxidase and Rho were down-regulated and nitric oxide synthase, myosin and tumor necrosis factor-α were up-regulated in hemocytes at 24 h post dsRNA treatment. After WSSV challenge, The mortality, WSSV copy number and expressions of WSSV immediate early genes (IE1, IE2, DNA polymerase, VP28) were both decreased but the apoptosis rate was increased in CRU-dsRNA-treated shrimps, indicating that WSSV may take advantage of crustin-like to benefit its replication. After silenced the crustin-like, the results of phagocytosis showed that the phagocytic rate of shrimp hemocytes on WSSV decreased significantly. In contrast, the absence of crustin-like in shrimps increased the mortality following V. alginolyticus challenge, indicating that crustin-like may play a positive role in the antibacterial process. The phagocytosis experiment showed there was a higher phagocytosis rate of hemocytes after CRU-dsRNA treatment. The result indicated that V. alginolyticus may be able to use crustin-like to avoid phagocytosis of shrimp hemocytes. These results further added to our understanding of the function of crustin-like peptide and also provided its potential role in innate immunity in shrimp.

Sun B ，Wang Z ，Zhu F 《-》